This invention relates to compounds containing a cleavable linker between a deliverable compound, e.g., a pharmaceutical agent, and a targeting moiety, e.g., a protein.
There are many diagnostic and therapeutic agents which suffer from non-selectivity of effect. Thus, cells not in need of a particular treatment are nevertheless exposed to the treating agent. Analogously, cells not intended to be the subject of a diagnosis, e.g., an imaging procedure such as radioimaging or MRI, are nevertheless subjected thereto, i.e., are imaged. As a result of this pervasive non-selectivity problem, much effort has been expended to enhance the selectivity of such pharmaceutical agents.
One technique often used is the binding of the non-selective pharmaceutical agent to another chemical moiety which is capable of targeting the resultant conjugate to a desired site. In most cases, the conjugate must also provide a means for cleaving the "active" agent from the targeting portion. This combination of features poses a unique biochemical problem: Not only must the conjugate provide a targeting moiety specific for a given site, but the cleavability must also be functional at that site and in such a manner that the agent retains its therapeutic or diagnostic capabilities.
In one general method, a protein such as a monoclonal antibody is attached to the pharmaceutical agent. The antibody is selected to be specific for the type of cells desired to be treated or diagnosed. The targeting portion has been attached to the pharmaceutical agent by a variety of linking groups, some of which are cleavable linkers.
The concept of selectively cleavable linkers has been proposed as a method to deliver free active drugs (as well as imaging agents) to tumors using antibody carriers. The method utilizes the ability of antibodies to localize and bind to tumor cells in vivo, due to the presence of tumor-specific antigens on the surface of many tumor cells. By coupling these tumor-specific antibodies to tumor-treating drugs via a selectively cleavable linker which is cleaved upon localization of the complex, releasing the free drug, the drug can be delivered specifically to the tumor cells, thereby reducing the side-effects of the often toxic tumor-treating drugs. One goal of the development of such selectively cleavable linkers is to provide a mechanism by which the drug is delivered free of any covalent modification, which may be critical since such modification may render the drug biologically inactive.
One physiological parameter which can be exploited for selective linker cleavage at the tumor site is pH. It has been reported that the pH of the extracellular fluid surrounding the tumor cells inside solid tumor masses is acidic (Tannock, I. F. et al., Cancer Research 49, 4373-4384 (1989)). In addition to extracellular pH, receptor mediated endocytosis of the antibody conjugate by a tumor cell followed by fusion of the endosome with a lysosome exposes the conjugate to an acidic pH of 4.5-5.0 (see, e.g., "The Pathway of Endocytosis, Pastan, I. and Willingham, M. C. in Endocytosis, I. Pastan and M. C. Willingham, eds. Plenum, N.Y. (1985)).
One of the more useful functional groups for derivatization by a linking reagent and which is available on some drugs, including, for example, the anticancer agents daunomycin and doxarubicin, is the amino group. One of the common methods to derivatize an amino group is to react it with a carboxylic acid group to form an amide bond.
However, typical amide bonds are notoriously resistant to non-enzymatic hydrolysis at acid pH. Harsh reaction conditions such as refluxing in concentrated aqueous acid for several hours to effect hydrolysis are not uncommon. On the other hand, certain amides have been shown to undergo facile hydrolysis under mildly acidic conditions. For example, the amide bond of maleamic acids (See Table 1) has been shown to be quite labile at pH 4.5 (See, e.g., Kirby, A. J. et al., J. Chem. Soc. Perkin II, 1206-1214 (1972) and Aldersley, M. F. et al., J. Chem. Soc. Perkin II, 1487-1495 (1974)). It has also been shown that by modifying the R.sub.a and R.sub.b groups, the rate of hydrolysis of the amide group can be substantially changed (Kirby and Aldersley, supra.). This relationship between structure and activity is summarized in Table 1:
TABLE 1 ______________________________________ ##STR1## Cpd R.sub.a R.sub.b R.sub.c, R.sub.d Relative Rate* ______________________________________ 1 H H Me, H 1 2 Me H Me, H 31.2 3 Et H Me, H 32.8 4 iPr H Me, H 44.5 5 tBu H Me, H 68.0 6 Me Me nPr, H 2.35 .times. 10.sup.4 7 (CH.sub.2).sub.4 Me, H 540 8 (CH.sub.2).sub.3 Me, H 4.2 .times. 10.sup.-5 9 (CH.sub.2).sub.2 Me, H 4.2 .times. 10.sup.-6 10 CHCHCHCH Me, H 0.72 11 H H iPr, H 0.7 12 H H tBu, H 2.6 13 H H Ph, H 4.5 14 H H Me, Me 42.7 N-methyl acrylamide (CH.sub.2 CHCONHMe) 1.4 .times. 10.sup.-7 (est.) ______________________________________
U.S. Pat. No. 4,569,789 (Blattler et al.) discloses the use of maleamic acids as acid-cleavable linkers. The linkers are formed by reacting the amine-containing compound with the maleamic acid portion of a linker which also contains a group which can react with a sulfur-containing group of a protein. These linkers are disclosed in the patent and the corresponding publication (Biochem. 24, 1517-1524 (1985)) as having a T.sub.1/2 for hydrolysis of the acid-cleavable bond of approximately 8 hr at pH 4.0. Unfortunately, a pH of 4.0 is at the lower end of the pH ranges most often cited in the literature for lysosomes and extracellular fluid surrounding tumor cells. Thus, the art still lacks an acid-cleavable linker which hydrolyzes at a physiologically more useful pH, e.g., 5.0-5.5, with a preferred T.sub.1/2 of 8 hr or, preferably, less.